Combined application of 17beta-estradiol and progesterone enhance vascular endothelial growth factor and surfactant protein expression in cultured embryonic lung cells of mice

Abstract

Preterm delivery is associated with disruption of the placental supply with 17beta-estradiol (E2) and progesterone (P). The aim is to evaluate the role of E2 and P on the regulation of key proteins in lung development in embryonic lung cells. Alveolar cell type II (AT-II) and central lung fibroblast cultures were established from mouse embryos. Cells were exposed for 24 hours to E2 and/or P, the estrogen receptor antagonist ICI 182.780 (ICI) and the progesterone receptor antagonist mifepristone (RU 486). The mRNA expression of vascular endothelial growth factor (VEGF) and surfactant protein B and C (SB-B, SB-C) was determined, and protein levels of VEGF were measured. Only the combined treatment with E2 and P increased mRNA expression and VEGF protein in AT-II cells and lung fibroblasts. Combined treatment also promoted SP-B and SP-C expression in AT-II cells. Pretreatment with ICI and RU 486 completely abolished the E2 and P induced effects. E2 and P enhanced expression of VEGF and surfactant proteins in primary embryonic lung cells and may be involved in regulating expression of key molecules for the prenatal lung development and postnatal lung function.

Figure 1

Alevolar cells type II (AT-II) and central lung fibroblasts stained with cytokeratin antibody. Note that AT-II stain positive for cytokeratin whereas fibroblasts do not. Therefore, contamination of AT-II cells in fibroblast cell cultures can be excluded.

Figure 2

Semiquantitative analysis of estrogen receptor alpha (ER-α), estrogen receptor beta (ER-β), and the progesterone receptor (PR) expression in central lung fibroblast and AT-II cell cultures. Note that all three hormone receptors are expressed in both cell cultures.

Figure 3

Quantitative analysis of VEGF gene expression in central lung fibroblasts treated for 48 hours with E2-8 M and P-8 M alone or in combination. Values were normalized against a housekeeping gene (HPRT) and expressed as % of controls. Note that only the combined application of both hormones significantly increased VEGF expression in central lung fibroblasts. Also note that the application of dexamethasone (D) had similar effects on VEGF expression. *P < .01 control versus E2/P-8 M, **P < .01 control versus D-8 M.

Figure 4

Quantitative analysis of VEGF gene expression in central lung fibroblasts treated for 48 hours with increasing concentrations of both E2 and P and with ICI/RU 486. Values were normalized against a housekeeping gene (HPRT) and expressed as % of controls. Note that the application of receptor antagonists 1 hour prior to hormone application (ICI/RU 486) antagonizes hormonal effects. P < .01 control versus E2/P-8 M; **P < .01 control versus E/P-6 M.

Figure 5

Quantification of VEGF protein release of lung fibroblasts treated for 48 hours with E2-8 M and P-8 M alone or in combination determined by ELISA. Note that corresponding with results obtained by gene expression analysis (Figure 3) VEGF protein is increased in central lung fibroblast cultures only by combined treatment with E2 and P. Pretreatment with ICI/RU 486 abrogated this effect. *P < .05 control versus E2/P-8 M.

Figure 6

Quantitative analysis of VEGF gene expression in alveolar cells type II treated for 48 hours with E2-8 M and P-8 M alone or in combination. Values were normalized against a housekeeping gene (HPRT) and expressed as % of controls. Note that only the combined application of both hormones significantly increased VEGF expression in central lung fibroblasts. Also note that the application on dexamethasone had similar effects on VEGF expression. *P < .01 control versus E2/P-8 M, **P < .01 control versus D-8 M.

Figure 7

Quantification of VEGF protein release of alveolar cells type II treated for 48 h with E2-8 M and P-8 M alone or in combination determined by ELISA. Note that corresponding with results obtained by gene expression analysis (Figure 6) VEGF protein is increased in alveolar type II cell cultures only by combined treatment with E2 and P. Pretreatment with ICI/RU 486 abrogated this effect. *P < .05 control versus E2/P-8 M.

Figure 8

Quantitative analysis of SP-B and SP-C gene expression in alveolar cells type II treated for 48 hours with E2-8 M/P-8 M in combination or with dexamethasone-8 M. Values were normalized against a housekeeping gene (HPRT) and expressed as % of controls. Note that combined hormonal treatment increased expression of both surfactant proteins to a similar extent like dexamethasone. Further note that pretreatment with the receptor antagonists ICI and RU 486 antagonized the hormonal effects. *P < .01 control versus E2/P-8 M; **P < .01 control versus D-8 M.